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71.
H. Yanase M. Okuda K. Kita Y. Sato K. Shibata Y. Sakai N. Kato 《Applied microbiology and biotechnology》1995,43(2):228-234
Dihydroxyacetoone synthase (EC 2.2.1.3), which is a key enzyme of the C1-compound-assimilating pathway in yeasts, catalyzes transketolation between formaldehyde and hydroxypyruvate, leading to the formation of dihydroxyacetone and CO2. When [13C]formaldehyde was used as a substrate with dihydroxyacytone synthase from Candida boidinii 2201, 13C was confirmed to be incorporated to the C-1 and C-3 positions of dihydroxyacetone, and the 13C content of each carbon (atoms/100 atoms) was estimated to be 50%. [13C]Methanol was also useful for the enrichment of dihydroxyacetone with 13C, when alcohol oxidase from a methylotrophic yeast was added for the conversion of methanol to formaldehyde. A fed-batch reaction with periodic addition of the substrates was required for the accumalation of 13C-labelled dihydroxyacetone at a higher concentration, because the enzyme system was relatively susceptible to the C donor, formaldehyde or methanol. The optimum conditions for the production gave 160mM (14.4 mg/ml) dihydroxyacetone for 180 min; the molar yield relative to methanol added was 80%. Diyhdroxyacetone kinase (EC 2.7.1.29) from methanol-grown Hansenula polymorpha CBS 4732 was a suitable enzyme for the phosphorylation of dihydroxyacytone. The phosphorylation system, comprising of dihydroxyacetone kinase, adenylate kinase, and ATP, could be coupled with the system for dihydroxyacetone production. A fed-batch reaction afforded 185 mM [1, 3-13C]dihydroxyacetone phosphate from [13C]methanol; the molar yield of the ester relative to methanol added was 92.5% 相似文献
72.
Electrophysiological properties of mouse bone marrow-derived mast cells (BMMC) were studied under the whole-cell clamp configuration. About one third of the cells were quiescent, but others expressed either inward or outward currents. Inwardly rectifying (IR) currents were predominant in 14% of the cells, and outwardly rectifying (OR) currents in 24%. The rest (22%) of the cells exhibited both inward and outward currents. The IR currents were eliminated by 1 mm Ba2+, and were partially inhibited by 100 μm quinidine. The reversal potential was dependent on extracellular K+, thereby indicating that K+ mediated the IR currents. The negative conductance region was seen at potentials positive to E K. The OR currents did not apparently depend on the extracellular K+ concentration, but were reduced by lowering the extracellular Cl? concentration. The OR currents were partially blocked by 1 mm Ba2+, and were further blocked by a Cl? channel blocker, 4,4′-diisothiocyano-2, 2′-stilbenedisulfonate (DIDS). In addition, the reversal potential of the OR currents was positively shifted by decreasing the ratio of external and internal Cl? concentrations, suggesting that Cl? was a major ion carrier. In cells exhibiting IR currents, the membrane potential varied among cells and tended to depolarize by elevating the external K+ concentration. In cells with OR currents, the resting potential was hyperpolarized in association with an increase in conductance. These results suggest that BMMC have a heterogeneous electrophysiological profile that may underlie a variety of ion channels expressed in different phenotypes of mast cells. Activities of both the inwardly rectifying K+ channel and the outwardly rectifying Cl? channel seem to contribute to the regulation of the membrane potential. 相似文献
73.
Changes in chorion proteins induced by the exudate released from the egg cortex at the time of fertilization in the teleost, Oryzias latipes 总被引:2,自引:2,他引:0
Takashi Iwamatsu Yasushi Shibata Toshihiro Kanie 《Development, growth & differentiation》1995,37(6):747-759
The chorion of unfertilized medaka Oryzias latipes eggs consists of two major proteins (77–73 and 49 kDa) and a minor 150 kDa protein. Upon fertilization, these major chorion proteins are polymerized to insoluble high molecular weight proteins via the temporary formation of several new proteins (132, 114, 62 and 61 kDa). Increasing chorion toughness is closely related to the formation of high molecular weight proteins and the increasing insolubility of the chorion proteins. The changes in chorion proteins and hardening could be induced in vitro in isolated chorions by an egg exudate, which includes cortical alveolar contents. The effects of temperature and pH on the egg exudate-induced changes in chorion proteins were examined in the present study. The major proteins could be digested by proteolytic enzymes. The 49 kDa protein was PAS-positive. Analysis with polyclonal antibodies against the major proteins demonstrated that the temporarily formed 62 and 61 kDa proteins were derived from the 77–73 kDa protein and that higher molecular weight proteins, newly formed in the process of chorion hardening, contained the same epitopes as did the 77–73 and 49 kDa proteins. The results suggest that the changes in chorion proteins of the medaka egg at the time of fertilization can be induced by an enzyme(s) released from the egg cortex into the perivitelline space. 相似文献
74.
Molecular Evolution of the Duplicated Amy Locus in the Drosophila Melanogaster Species Subgroup: Concerted Evolution Only in the Coding Region and an Excess of Nonsynonymous Substitutions in Speciation
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From the analysis of restriction maps of the Amy region in eight sibling species belonging to the Drosophila melanogaster species subgroup, we herein show that the patterns of duplication of the Amy gene are almost the same in all species. This indicates that duplication occurred before speciation within this species subgroup. From the nucleotide sequence data, we show a strong within-species similarity between the duplicated loci in the Amy coding region. This is in contrast to a strong similarity in the 5' and 3' flanking regions within each locus (proximal or distal) throughout the species subgroup. This means that concerted evolution occurred only in the Amy coding region and that differentiated evolution between the duplication occurred in the flanking regions. Moreover, when comparing the species, we also found a significant excess of nonsynonymous substitutions. In particular, all the fixed substitutions specific to D. erecta were found to be nonsynonymous. We thus conclude that adaptive protein evolution occurred in the lineage of D. erecta that is a ``specialist' species for host plants and probably also occurs in the process of speciation in general. 相似文献
75.
Isolation and characterization of Enterobacter cloacae mutants which are defective in chemotaxis toward inorganic phosphate. 总被引:1,自引:0,他引:1
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Enterobacter cloacae IFO3320 is attracted to Pi when cells are starved for Pi. Two Tn1737KH-induced mutants, which were constitutive for alkaline phosphatase, failed to exhibit Pi taxis even under conditions of Pi limitation. Both of the mutant strains exhibited normal chemotactic responses to peptone, suggesting that they are specifically defective in Pi taxis. Cloning and sequence analysis showed that the TN1737KH insertions were located in either the pstA or pstB genes which encode the channel-forming proteins of the Pi-specific transport (Pst) system in E. cloacae. These results suggest that the E. cloacae Pst system is required for Pi chemoreception. 相似文献
76.
Yasushi Isashiki Norio Ohba Toyoko Yanagita Naoko Hokita Norihito Doi Masanori Nakagawa Masayuki Ozawa Noriko Kuroda 《Human genetics》1995,95(1):105-108
We have identified a new mutation of Norrie disease (ND) gene in two Japanese males from unrelated families; they showed typical ocular features of ND but no mental retardation or hearing impairment. A mutation was found in both patients at the initation codon of exon 2 of the ND gene (ATG to GTG), with otherwise normal nucleotide sequences. Their mothers had the normal and mutant types of the gene, which was expected for heterozygotes of the disease. The mutation of the initiation codon would cause the failure of ND gene expression or a defect in translation thereby truncating the amino terminus of ND protein. In view of the rarity and marked heterogeneity of mutations in the ND gene, the present apparently unrelated Japanese families who have lived in the same area for over two centuries presumably share the origin of the mutation. 相似文献
77.
The substrate specificity of rice embryo benzoyl-L-argininep-nitroanilide hydrolase (BAPAase) was examined. No endopeptidaseactivity toward protein substrates was detectable. Small peptides(less than 8 residues) and amide, ester substrates, however,were hydrolyzed very well at the carboxyl side of the lysineor arginine residue. No other peptide bond was hydrolyzed. TheN-terminal arginine of the substrates was released very slowly.Peptides with lysine or arginine penultimate to the C-terminalposition were hydrolyzed well and released an amino acid. Theoxidized insulin B chain (30 residues) was cleaved very slowlyat the C-terminal Lys-Ala bond, whereas an Arg-Gly bond at aninner position was not cleaved. The hydrolytic rate increasedafter the chain length was shortened by chymotryptic digestion.These results show that the rice embryo BAPAase is a novel enzymewhich has mixed endopeptidase-carboxypeptidase activity towardthe Arg-X and Lys-X bonds of small peptides, a characteristicintermediate between trypsin and serine carboxypeptidase. Thisenzyme may act in the breakdown of small peptides that havephysiological functions. (Received May 26, 1984; Accepted August 29, 1984) 相似文献
78.
Benzoyl-L-arginine p-nitroanilide hydrolase (BAPAase), whichhas both endopeptidase and carboxypeptidase activity towardthe Arg-X or Lys-X bond of small peptides [Shibata and Doi (1984)Plant & Cell Physiol. 25: 1421], was purified from riceembryos by ammonium sulfate and polymin fractionations and byion exchange, gel exclusion and hydrophobic chromatographies.The purified enzyme was homogeneous when analyzed by polyacrylamidegel electrophoresis. It was unstable in the absence of surface-activereagents such as Triton X-100. Maximum activity for benzoyl-Largininep-nitroanilide (L-BAPA) or carboxypeptidase activity towardbutoxycarbonyl-Gly-Lys-Leu was obtained at pH 9.0. L-BAPA athigh concentrations inhibited the enzyme's activity. Di-isopropylphosphofluoridate, N-tosyl-L-lysine chloromethyl ketone, leupeptinand antipain, which are specific inhibitors of trypsin, inhibitedBAPAase activity, but soybean and rice bran trypsin inhibitorhad no effect on it. Sulfhydryl reagents strongly inhibitedthe BAPAase activity. (Received May 26, 1984; Accepted August 29, 1984) 相似文献
79.
A rapid method is described for the preparation of serum alpha1-acid glycoprotein from rats with inflammation induced with turpentine oil injection. The protein obtained by two purification steps, batchwise adsorption with DEAE-cellulose followed by chromatography on CM-cellulose, was proved to be native alpha1-acid glycoprotein in a high degree of purity by electrophoretical, immunological, ultracentrifugal and carbohydrate analysis. The monospecific and potent antiserum to this protein was prepared by immunizing rabbits with the desialyzed material emulsified with Freund's incomplete adjuvant. Using purified alpha1-acid glycoprotein and its specific antiserum, the concentration of alpha1-acid glycoprotein in rat serum was determined by single radial immunodiffusion. Abnormally high levels of its concentration (5-6 times higher than the control) were observed in inflammatory and tumor bearing rats. 相似文献
80.
Specific antisera against somatostatin-28 were prepared by absorption of somatostatin-28 antisera with sepharose 4B-somatostatin-14. Indirect immunofluorescence techniques using somatostatin-14 antisera and specific antisera against somatostatin-28 were carried out to elucidate the time of occurrence of somatostatin-28 in the fetal pancreatic islets and to ascertain whether somatostatin-28 was present in the adult pancreatic islets or not, and further to examine whether cells reacting with specific antisera against somatostatin-28 are identical to those reacting with somatostatin-14 antisera or not. Somatostatin-28 like immunoreactivity occurred in the fetal pancreatic islets at 11th week's gestation and was found in all fetal pancreatic islets examined in the present study. It was also found in the adult pancreatic islets. Furthermore, cells reacting with specific antisera against somatostatin-28 in the fetal and adult pancreatic islets were identical to those reacting with somatostatin-14 antisera. Thus, the present study elucidated the presence of somatostatin-28 like immunoreactivity in the human pancreas. However, it could not be decided whether cells reacting with somatostatin-28 antisera contain either only somatostatin-28 or both somatostatin-28 and somatostatin-14; in other words, whether somatostatin-14 is produced from somatostatin-28 or not, since somatostatin-14 antisera had a cross-reactivity to both somatostatin-14 and somatostatin-28. 相似文献